A newly introduced salt bridge cluster improves structural and biophysical properties of de novo TIM barrels

Abstract

Protein stability can be fine-tuned by modifying different structural features such as hydrogen-bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels. The salt bridge variants exhibit similar thermostability in comparison with their parental designs but important differences in the conformational stability at 25°C can be observed such as a highly stabilizing effect for two of the proteins but a destabilizing effect to the third. Analysis of the formed geometries of the salt bridge cluster in the crystal structures show either highly ordered salt bridge clusters or only single salt bridges. Rosetta modeling of the salt bridge clusters results in a good prediction of the tendency on stability changes but not the geometries observed in the three-dimensional structures. The results show that despite the similarities in protein fold, the salt bridge clusters differently influence the structural and stability properties of the de novo TIM barrel variants depending on the structural background where they are introduced.

Keywords: (β/α)8 barrel; DeNovoTIMs; TIM barrel; de novo protein design; protein folding; protein stability; salt bridge cluster.

FIGURE 1

Strategy to introduce a salt bridge cluster in de novo TIM barrels. Crystal structure of sTIM11noCys is shown with residues 20, 66, 112, and 158 highlighted as sticks, which were used for the introduction of the salt bridge cluster. It was added in the de novo TIM barrels replacing Q20 and Q112, belonging to the first and third quarters, by arginine residues, and Q66 and Q158, from the second and fourth quarters, by glutamic acid residues

FIGURE 2

Folding stability of the salt bridge variants. (a) Thermal unfolding experiments followed by DSC. Endotherms were collected at 1.5°C min⁻¹ and protein concentration of 1.0 mg ml⁻¹. Dotted lines show the parental proteins and continuous lines the salt bridge cluster variants. (b) Chemical unfolding with urea at 25°C, circles representing CD data and triangles fluorescence data. Dotted and continuous lines represent the fitting of the data to a reversible two‐state model for the parental and salt bridge variants, respectively. Data from sTIM11noCys, DeNovoTIM6, and DeNovoTIM13 are reported in Reference . All experiments were collected in 10 mM sodium phosphate pH 8

FIGURE 3

Stability curves of the salt bridge variants. Curves were constructed using the parameters from DSC experiments and the Gibbs–Helmholtz equation. Open symbols indicate the ΔG value at 25°C determined by chemical unfolding. Data from sTIM11noCys and DeNovoTIM6 are reported in Reference

FIGURE 4

Structural conformations of the salt bridge interactions in the de novo TIM barrels. (a) sTIM11noCys‐SB (crystal form 1, PDB ID: 7OSU). (b) DeNovoTIM6‐SB (crystal form 1, PDB ID: 7OSV). (c) DeNovoTIM13‐SB (PDB ID: 7P12). In all panels, upper figures indicate the view from the bottom of the barrel with the salt bridge residues highlighted in sticks. 2Fo–Fc electron density maps contoured at 1σ are shown as a gray mesh for all the residues/water involved in the salt bridge cluster. Lower figures show the side view of the salt bridge interactions to analyze their planarity. Dotted lines indicate the salt bridge interactions between the mutated residues whose measures are reported in Table 2